6520471Broad Institute2120-05_Inhibitor_Dose_CherryPick_ActivityCell-based secondary assay to test the inhibitory activity of small molecule on Plasmodium flaciparum (3D7 strain) survival in red blood cells Measured in Cell-Based System Using Plate Reader - 2120-05_Inhibitor_Dose_CherryPick_ActivityKeywords: Plasmodium falciparum, malaria, Hsp40Assay Overview: This assay has been performed in collaboration with Dr. Amanda Lukens from Dyane Wirth's laboratory at the Harvard School of Public Health, Boston MA. Plasmodium falciparum has the capacity to infect red bllod cells and use the transcriptional and translational machinery in the host cell to replicate. In the assay, we are testing the ability of a small molecule to kill the parasite Plasmodium falciparum 3D7 strain present inside red blood cells. The viability of the parasite was determined by measuring the fluorescence emitted by the interacton the green-based fluorescent dye SYBR with the parasite DNA content detect present inside the host cells. Mefloquine, a known antibiotic able to Plasmodium falciparum, was used as positive control.Expected Outcome: Compounds decreasing the fluorescent signal normalized to the positive control more than 35% (AbsACNN-35) at a concentration below 10uM will be considered actives.SYBR Drug Assay Standard Protocol Plasmodium falciparum 3D7 strainGeneral notes:- cultures are maintained and the assay performed in RPMI media supplemented with Albumax II (recipe below)- stock cultures are maintained at 5% hematocrit- cultures are maintained in incubators at 37 degrees C and 4.1% CO2 + 1%O2 + balance N2 gas environmentDay -2 Synchronize cultures for assay1. Make Giemsa slide of seed culture(s) and determine total parasitemia and ring parasitemia- cultures that are minority rings are difficult to effectively synchronize- cultures >3 days post-thaw can be used; don't use cultures > 8 weeks from thaw date2. Calculate volume of culture to synchronize according to percentage of rings and hematocrit3. Set up a 0.75-1% ring culture of appropriate volume- don't use blood older than three weeks or culture medium thawed for more than a week4. Synchronize parasites- pellet RBCs by centrifugation- remove supernatant and resuspend in 40% original culture volume of 5% pre-warmed sorbitol solution (i.e. for a 25mL culture, add 10mL sorbitol)- incubate cells in sorbitol at 37 degrees C or room temperature for 10-20 minutes- centrifuge cells and remove sorbitol containing supernatant- resuspend cells in an appropriate volume of complete media for a 5% hematocrit culture and return to incubatorDay -1 Feed cultures1.Make a slide of each synchronized culture to check synchronization and parasitemia- if trophozoite parasitemia is >2%, split culture to 1% trophs and replace media; otherwise, just replace media on culture2. Make sure sufficient blood and complete media are available for assay tomorrow3. Calculate drug dilutions, label plates and organize equipment for assay tomorrowDay 0 Set up assay1. Feed cultures on morning of assay; prepare and warm media for assay2.Prepare Plate Map and HTS Excel files for assay3.Make Giemsa slides and determine total and ring parasitemias- parasitemias should be between 2-7% with 90% of parasites at ring stage4. Harvest 5mL sample for RNA- centrifuge to obtain red cell pellet- lyse RBCs with 0.15% saponin solution- transfer lysate to a microfuge tube and centrifuge at max speed for 5 minutes- remove supernatant and resuspend pellet in 250uL of buffer RTL from Qiagen RNAeasy Mini Kit- store sample at -80 degrees C for later RNA extraction5. Determine hematocrit of culture6. Thaw compounds and prepare 96-well drug dilution and control plates7. Transfer drug dilutions with Velocity robot to 384 well plates8. Make cell suspension for dispensing into assay plates- prepare a 1% parasitemia/1.3% hematocrit solution for the assay plates (final = 1% parasitemia/1%hematocrit in assay plate)PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.EXPECTED OUTCOME: Active compounds result in decreasing readout signal.ACTIVE CONCENTRATION LIMIT:For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).NORMALIZATION:The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.Experimental wells values were scaled to this range.PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold -35.0AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).AC values were calculated up to the active concentration limit described for each sample.pAC was set to equal -1*log10(AC)PUBCHEM_ACTIVITY_OUTCOME:Activity_Outcome = 1 (inactive) when:a) compound shows activity but in a direction opposite to the expected outcomein these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to nullb) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, orc) AC > AC_limitActivity_Outcome = 2 (active) when:AC <= AC_limitActivity_Outcome = 3 (inconclusive) when:a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, orb) The fit was deemed not valid due to poor fit quality.PUBCHEM_ACTIVITY_SCORE:If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),then PUBCHEM_ACTIVITY_SCORE = 0If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)Scores relate to AC in this manner:120 = 1 pM90 = 1 nM60 = 1 uM30 = 1 mM0 = 1 MWhen the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.Note:The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.504552Summary assay36329Plasmodium falciparum 3D7124809271Plasmodium falciparum HSP40 (malaria), Subfamily Ahttp://www.broadinstitute.orgBroad Research Institute1AbsAC35_Qualifier<, =, or >42542AbsAC35_uMThe concentration at which the fitted curve passes activity threshold 35.153pAbsAC35_MEqual to -1*log10(AbsAC35).12544Hill_SlopeThe slope at AC5012545S0_(%)The fitted activity value at zero concentration1156Sinf_(%)The fitted activity value at infinite concentration1157Num_PointsThe number of data points used to generate the plot22548Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentration1159Max_Activity_Conc_uMThe concentration at which the maximum activity is observed1510Max_Concentration_uMMaximum valid test concentration1511Activity_at_0.0235uM_(%)The average measured activity of all accepted replicates at the specified concentration1150.02349999919533735112Activity_at_0.068uM_(%)The average measured activity of all accepted replicates at the specified concentration1150.0680000036954885113Activity_at_0.21uM_(%)The average measured activity of all accepted replicates at the specified concentration1150.2099999934434895114Activity_at_0.6uM_(%)The average measured activity of all accepted replicates at the specified concentration1150.6000000238418585115Activity_at_1.8uM_(%)The average measured activity of all accepted replicates at the specified concentration1151.799999952316285116Activity_at_5.6uM_(%)The average measured activity of all accepted replicates at the specified concentration1155.599999904632575117Activity_at_16uM_(%)The average measured activity of all accepted replicates at the specified concentration115165118Activity_at_50uM_(%)The average measured activity of all accepted replicates at the specified concentration1155051121CR Plot Labels 1ConcentrationResponse01 R03 DA031664-01284409812481=217.5234.75642.415-9.4816-1007168-84.771950105011-8.87912-11.41513-9.54914-6.92515-10.51416-12.06317-31.07618-84.771201321684490911050.32880756258964560.328807562589645716849.49190.023510501149.491126.31813-4.54314-8.49715-11.55716-7.61917-6.32718-14.63420132168452781105-4.318622589111336-4.318622589111337168-8.49690.6105011-5.04212-5.39113-7.6914-8.49615-6.54116-7.17172.799182.981201321684583512451=234.6834.4641.21256.726-1007158-44.4949501050118.66125.553135.175149.979153.43116-5.3817-10.28218-44.49420132168521611105-6.652453422546396-6.652453422546397158-23.55295010501110.51212-12.60313-3.24814-8.89715-10.35416-6.26217-7.26518-23.552201321685836911050.65695142745971760.656951427459717714816.5391.8105011-6.135123.21513-14.73145.991516.53164.73717-1.28418-2.824201321686144511052.0336735248565762.03367352485657716831.46590.023510501131.465126.37613-2.5714-3.31315-8.507160.26617-4.22418-3.2242013216371144312561=22.3835.62342.22653.6666-106.7457158-105.389161050117.922124.34913-2.675142.68915-21.60216-82.46517-105.3818-105.3572013216371164412451=230.7434.51242.1375-12.0196-1007168-55.901950105011-3.98912-14.40713-12.86314-10.77715-17.06416-14.35317-18.5318-55.9012013216371228312491=212.4134.906445-10.2626-1007168-104.789501050117.95712-16.24413-20.47114-12.30215-11.05816-9.69817-56.34818-104.78201321637160221105-3.93031382560736-3.93031382560737168-9.37790.068105011-5.09212-9.37713-5.89614-4.22915-2.75416-6.025177.42118-5.4882013216371718612481=215.1534.82445-10.1546-1007158-102.999950105011-10.48212-12.60213-10.64814-7.5515-13.62216-8.64717-38.2718-102.9992013216424563012461=225.3634.59642.8754.5726-1007158-80.18395010501113.13712-0.893130.4011411.09515-4.03164.11817-10.02318-80.1832013216424692512501=29.835.00943.694517.7486-105.327168-104.999501050115.3031215.0511322.2531419.3721526.903166.71317-83.21818-104.992013216425549812481=215.1234.8240.50755.9826-1007168-62.17795010501117.96712-4.18713-7.06514-13.07415-15.99816-18.97817-21.35418-62.1772013216425576112481=217.7934.7544523.4186-1007168-105.39795010501122.9711222.8651328.1721423.7881523.911617.43917-21.20618-105.3972013216425586512421=270.0434.15540.55256-257158-16.54495010501131.911215.037132.735149.256154.654161.00117-6.05118-16.5442013216425917311055.2545981407165565.25459814071655716812.2599161050115.302127.258131.598147.358152.01116-1.4151712.259187.6672013216426086012501=210.7634.96843.6275-4.1476-1007168-103.649501050114.04412-8.30313-1.90114-4.35515-9.66916-8.94717-67.76918-103.6420132164262306110514.8891878128052614.8891878128052715827.3469501050113.997125.5931310.9281411.9881514.6621625.4651724.4231827.3462013216426388811059.2519483566284269.25194835662842715814.4379161050114.4211213.95138.479148.373156.056169.1741714.437186.71201321679679321105-4.443903446197516-4.44390344619751715817.059950105011-4.70212-4.617131.21714-12.99915-13.06216-16.8217-5.9051817.0592013216797124912461=223.0234.6384454.3876-1007168-103.414950105011-3.73812-5.639131.698149.253154.0421616.79117-1.70718-103.4142013216797204912481=216.1334.79244524.6116-1007168-104.34195010501123.8391216.4851318.5291427.3051532.8151627.46817-33.81818-104.3412013216797307112571=22.0235.6954451.7466-106.8227158-106.333950105011-0.64126.89413-1.007140.50915-23.62616-106.03417-105.11518-106.3332013216797515512511=28.5635.06841.128522.1796-1007168-103.66695010501135.1521227.9831314.556146.56315-3.2616-15.9517-44.14318-103.6662013216797586812541=24.3235.36542.25352.16-104.3227158-106.78295010501112.094123.57913-4.55514-2.696150.33916-52.77217-90.21318-106.7822013216797837812671=20.18236.7442.15851.3336-106.1287158-105.92395.61050111.52912-5.91413-41.80714-92.39915-105.37716-105.92317-105.66618-105.7820132161153288212631=20.44836.34942.003523.546-108.2787138-106.42895010501122.1711228.72313-0.49914-50.32515-105.60916-104.40918-106.42820132161472152312481=217.7134.752445-11.0126-1007158-102.654950105011-16.45112-12.24513-12.66614-8.12515-8.84516-11.86817-26.61118-102.65420132161472328612471=222.2734.6524450.5336-1007158-101.2249501050116.10512-0.09413-1.458140.98815-0.69916-2.15417-7.25718-101.22420132161472475811040.886506-307148-26.44895010501120.2912-0.716130.89814-1.02715-9.94316-16.36817-17.05618-26.4482013216147248131105-17.59855651855476-17.59855651855477168-49.179950105011-17.01112-16.95613-12.69214-6.86915-11.53116-11.62217-14.92818-49.17920132161472910012441=241.8734.3784457.826-1007168-54.52495010501110.9691215.285133.862141.363156.927165.379179.99218-54.52420132161472946412591=21.1335.9474454.2356-105.5047158-105.9279501050113.879123.041134.659140.86815-82.13616-105.48117-104.55218-105.92720132161472962712441=242.634.37140.55256-1007158-24.97495010501160.2251220.9051313.629146.30315-0.1816-2.937170.17518-24.97420132161472965312481=216.934.772445-5.1676-1007168-105.211950105011-3.262128.47413-1.94314-6.43815-13.78216-15.39317-29.18218-105.211201321614730163110513.2988653182983613.2988653182983714830.01891610501110.838124.624139.3141411.5751516.6391614.1921730.0181819.222013216147303781105-8.613192558288576-8.613192558288577168-31.054950105011-9.13812-8.31113-9.82814-1.94515-4.597161.46617-5.49918-31.05420132161473038512551=23.5535.4543.1275256-106.847168-107.12695010501123.6761222.7661326.2331427.2371512.86316-77.45117-105.11118-107.12620132161473127812571=21.9135.7194451.2626-106.6787158-105.9819501050115.58112-4.124130.014143.06915-28.65416-105.71217-105.92918-105.98120132161473336812591=21.2135.917445-9.2816-103.527168-103.444950105011-7.70512-7.27113-13.12914-11.06215-70.20516-103.29117-103.26118-103.44420132161473589412501=210.3834.98442.0751.8366-1007168-93.1199501050116.20212-3.5713-3.685145.602153.60716-12.81817-57.27618-93.11920132161473612812471=222.2534.6534458.1496-1007158-96.71195010501126.6131213.526137.036144.163155.065161.46917-4.26818-96.71120132161473917212481=214.1634.8494451.396-1007168-104.603950105011-2.037120.173131.151143.566152.071162.69817-46.65618-104.60320132161473962212481=217.3934.764456.1496-1007168-107.029501050114.831120.182133.755148.573159.24169.12317-25.14818-107.0220132161474000112481=216.1734.79144514.9636-1007168-103.85495010501110.166126.7121311.2791415.3321525.6131620.13117-33.22918-103.85420132161474196612551=23.2335.49142.0715-6.2226-112.1767168-109.082950105011-3.79712-5.51213-7.67114-8.03115-18.76916-61.60317-105.83218-109.08220132161474251912551=22.9135.53642.8535-0.2026-106.7547168-107.45495010501113.11112-0.87713-12.61714-1.23915-11.61516-81.18517-104.18418-107.45420132161474678212521=26.6935.175445256-1007128-104.97895010501127.4781227.486137.58149.8371554.7316-13.32117-100.22918-104.97820132161474694612441=235.634.44942.0495-2.5846-1007158-43.248950105011-4.59712-0.58813-0.91214-6.10415-8.04161.08917-13.16618-43.24820132161474699612621=20.66336.17844513.0156-106.6347128-106.3695010501113.8861211.0331313.32814-23.83615-103.95918-106.362013216173867051105-18.49246597290046-18.49246597290047168-60.209916105011-7.15212-7.90713-6.43914-13.93415-19.07716-18.14117-60.20918-15.08120132161738713012441=241.1334.38644512.7626-1007138-59.3339501050114.43412-0.1211321.9911415.231154.44169.8251722.34818-59.3332013216173880111105-15.05212974548346-15.05212974548347168-29.761950105011-12.64912-10.21613-10.65814-11.45415-7.45616-9.83617-28.38718-29.761201321617388195110528.1338748931885628.1338748931885716874.02690.2110501121.3571238.4911374.0261423.5251536.8341615.8971716.89518-1.95420132161738847512471=219.1134.71942.3575-14.6856-1007168-79.097950105011-8.08612-15.77313-15.45914-15.18315-15.66916-20.28817-28.26518-79.09720132161738860312481=216.4334.784445256-1007168-98.06695010501133.851219.3321330.8931442.3481526.2011635.00717-32.01918-98.06620132161740777412541=23.635.44441.0185-13.2586-1007158-84.85950105011-12.30812-12.17613-19.35114-16.40415-24.54716-43.67217-66.11618-84.8520132161741053512441=240.2434.39542.61851.8126-1007148-40.1529501050112.074124.221135.94414-0.246151.94416-1.62717-1.00318-40.152201321617410955110510.1873254776001610.1873254776001715815.54791.810501112.639127.77139.577146.9511515.547163.0541713.531814.67420132161741102012451=228.6534.543445-14.066-1007158-81.189950105011-13.47712-18.86813-15.36414-10.03415-16.98316-14.80417-10.49818-81.18920132161741163812591=21.235.92142.72150.7766-106.6317168-107.1959501050114.804123.1613-6.73414-6.19815-63.85916-104.20617-105.20718-107.19520132161741200212511=28.7735.05742.98951.8166-1007168-103.8469501050111.068120.68513-1.258141.099157.77316-12.34217-76.1918-103.84620132161741336912451=229.6234.52843.385-15.5796-1007168-69.317950105011-12.89712-20.44213-17.20214-15.49515-15.98916-11.29617-18.86718-69.31720132161741347111053.1883401870727563.18834018707275713813.086916101611-6.39112-0.59133.199144.367152.582166.6551713.08620132161741358512451=228.3334.54842.04358.9496-1007168-66.02995010501114.4131214.877137.186145.491155.808161.72617-8.37218-66.02920132161741370512461=222.6834.64441.43652.1386-1007158-64.7795010501112.409121.50913-1.138142.85715-6.47116-7.68617-17.59418-64.7720132161741522912441=237.2834.42941.37858.3746-1007158-38.47495010501145.616129.85213-6.065143.757153.993167.37217-6.49218-38.4742013216174156121105-1.944141149520876-1.944141149520877158-27.302950105011-1.7612-2.27113-3.49214-2.161151.662165.847171.24518-27.30220132161743191512521=26.2935.2014459.7946-102.1267168-104.3991610501119.597128.894139.654144.751154.92616-21.62217-104.3918-96.87620132161743297912491=212.7134.89643.55-10.0976-1007158-104.426950105011-5.81112-7.92613-10.27314-11.4815-8.83616-18.89917-50.46718-104.42620132161743335812471=220.0234.699445-1.9146-1007168-105.737950105011-6.723121.21413-4.582141.05315-4.24116-0.6917-14.98918-105.7372013216174334971105-5.444264411926276-5.444264411926277168-19.985950105011-1.6712-0.44113-0.4414-1.44415-4.17116-8.13317-7.2718-19.98520132161743380712531=25.5535.25642.67455.0086-1007158-103.87895010501122.2741213.521135.23614-1.62915-10.97816-32.31517-95.61218-103.87820132161750515112481=215.7134.8044454.3076-1007168-106.9469501050112.028126.127133.726144.162157.621161.35217-35.48518-106.94620132161750729312481=215.734.80443.0635-15.7816-1007168-93.504950105011-6.55812-12.69213-19.14514-18.08715-16.24816-23.71817-34.97718-93.50420132161750741312521=26.135.21542.36458.1776-106.2897168-103.76995010501114.839129.168134.052149.36815-1.79416-27.817-91.63718-103.76920132161750762212541=23.9935.3994453.4376-105.7457168-105.4869501050115.485120.03131.605142.326155.98616-71.55317-104.96818-105.48620132161750887712481=214.7234.832445-4.3196-1007168-104.194950105011-312-11.74713-7.078143.69815-0.73316-8.11717-41.23518-104.19420132161750905512611=20.8236.08642.5945-2.6356-104.8517168-104.6279501050117.39812-11.2913-5.75214-19.75815-82.48316-104.32417-104.01918-104.62720132161751115412521=25.7135.24343.1535-0.646-1007158-104.607950105011-6.289124.36313-0.951140.91515-2.67616-33.6317-92.84218-104.60720132161751270612481=214.6734.83442.26258.5336-1007168-86.42395010501111.145124.229134.034146.4651512.855165.62717-42.95118-86.42320132161751279012441=240.6634.391445-6.5656-1007158-44.633950105011-4.04112-8.0713-11.70914-6.72715-6.24816-7.39317-2.92118-44.63320132161751288012601=21.0635.97542.484510.9616-107.0297158-106.54291610501112.2341210.536137.97214-5.22815-71.91316-106.05217-106.54218-105.67620132161751575612551=23.4635.46142.9915-4.8196-106.6597168-105.9329501050118.4512-13.3713-10.79714-3.47815-10.91416-69.81917-104.97218-105.93220132161751726112481=217.1834.7654454.1666-1007168-108.6479501050114.958120.636137.1341410.162151.23716-0.53717-26.36418-108.64720132162240304612601=20.89636.0484453.2386-106.2027168-105.68395.61050111.677122.644132.85214-3.34815-98.93316-105.68317-105.37318-105.16220132162240525712521=26.5735.18241.2425256-1007168-104.2695010501141.9791230.8421316.761411.428150.24816-18.05417-65.89218-104.2620132162240532412511=27.9135.102445-4.3766-107.2787168-105.932950105011-6.34812-3.33613-6.98214-2.45915-4.56916-11.52217-96.80118-105.93220132162240622711050.38529691100120560.38529691100120571587.821916105011-4.094122.124130.57140.23415-0.8316-0.348177.82118-2.46920132162240642712521=25.6935.24541.33959.0336-1007168-100.55695010501119.8351213.0513-3.536143.24515-8.08116-31.71417-68.19318-100.55620132162240676412491=211.9734.92243.263520.3286-1007168-107.39595010501140.4741220.6071315.0591417.959158.0171611.38217-61.50318-107.39520132162240840412471=218.3334.73741.3565-5.7456-1007168-66.261950105011-3.01312-12.96513-4.64114-2.46615-6.15916-16.42317-29.97618-66.26120132162240856812641=20.42236.37541.3156.7916-105.177168-103.8499501050114.853121.76413-15.13914-47.80115-81.82716-100.53217-103.83818-103.84920132162241005212511=27.7735.114455.0696-110.3677158-106.511916105011-6.512125.835137.975145.904152.42716-3.51417-106.51118-105.31620132162241030512561=22.3435.63141.143518.1696-1007168-105.82195010501133.2711214.573134.28214-13.96515-16.94516-53.64417-102.58818-105.82120132162241095912441=242.634.37140.55256-1007158-18.1495010501133.7671219.0031310.281147.1561510.896160.77117-2.25218-18.1420132162241150112531=24.9635.3054451.0066-109.6927158-110.3669501050111.81112-5.14130.096144.229153.74116-47.59217-107.27718-110.36620132162241205212471=220.2234.69443.5515-1.6596-1007158-93.17195010501114.67312-3.349133.90314-1.32415-10.31116-6.15217-19.32218-93.17120132162241324612491=212.8234.89240.82256.6126-1007168-62.095950105011-4.325129.644136.2331410.54815-8.33216-21.60117-40.52518-62.09520132162241468612521=26.9835.156445-10.6616-105.2827168-104.5989501050110.35912-9.92513-17.94214-14.60815-11.85916-21.07817-97.7918-104.59820132162241501212551=23.2735.48544517.4926-106.6287138-104.76391610161114.4751211.4361325.5411413.2881514.50916-92.5217-104.7632013216224154761105-17.39033889770516-17.39033889770517138-23.31891.8105011-14.7612-21.69213-3.68114-10.53815-23.31816-16.54717-19.09918-21.02420132162241594112531=25.3435.27241.895516.8196-105.2127168-105.23395010501116.407128.0161319.8191419.63159.56616-40.76317-83.68618-105.23320132162477960712451=228.7134.54243.20755.4946-1007168-77.4789501050116.586129.52136.282141.041152.379166.92417-3.8418-77.47820132162477965012471=22034.69943.84351.046-1007148-95.4819501050114.725121.056133.77514-0.90615-0.84616-3.08617-18.01318-95.48120132162478127712441=238.8434.41140.5522.2866-1007158-28.35695010501123.41212.189136.2841415.50915-0.73516-5.97217-11.98118-28.35620132162478641612501=29.4535.02542.7065-5.0186-1007158-103.803950105011-7.17212-2.58513-4.22514-5.22415-4.74316-16.31317-66.15318-103.80320132162478648312541=23.8235.41842.165-19.6726-1007168-103.6950105011-7.85612-20.10213-18.25814-25.43615-33.08416-43.98117-89.51318-103.620132162478889412561=22.3935.62243.19159.5166-107.2217168-106.8919501050119.9791221.301138.01914-2.98415-12.67516-96.88317-106.33218-106.8912013216247893361105-6.693516254425056-6.693516254425057158-28.04950105011-4.63212-8.46613-7.4614-7.008150.10416-1.36417-7.35518-28.0420132162478966212561=22.7735.55842.6455256-107.5847168-107.01895010501151.341225.1081318.6421423.36715-1.77616-86.69517-106.44918-107.0182013216247903441105-0.4426948726177226-0.4426948726177227158-78.6469501050110.316124.094132.41144.226156.341167.1241711.4918-78.64620132162479285412481=216.4734.78342.31557.8916-1007168-91.3289501050118.5711210.4651312.56146.859153.96516-2.32417-31.24118-91.32820132162479391312521=25.6435.24941.2835-2.4756-52.4757168-46.813950105011-6.805121.22613-11.78314-3.27315-17.08116-35.14117-46.28918-46.81320132162479393912451=234.2834.46542.24559.9486-1007158-57.9379501050110.435128.3261311.3841413.0971517.812167.12517-2.1518-57.9372013216247946891105-1.682875514030466-1.682875514030467168-6.0139161050112.48712-1.22613-1.17314-3.50815-1.88116-3.19517-6.013181.04620132162479485612451=228.7334.54240.5514.2756-1007138-39.61495010501120.228122.70213-2.622143.90615-5.23216-15.41417-18.5918-39.61420132162479489612471=220.7634.68343.0345-6.1826-1007168-87.148950105011-11.64612-8.30213-9.61114-0.87915-2.19116-5.06817-22.12418-87.14820132162479702712501=210.1134.9954453.7176-1007158-106.26395010501126.72123.69713-0.5714-0.77715-0.14116-0.11117-78.90818-106.26320132162479742312481=215.8634.8445256-1007128-106.83795010501128.9851223.8081326.2281449.631528.88717-35.5918-106.83720132162479759712491=213.8634.85843.0195-1.5246-1007168-100.661950105011-6.25812-1.156131.711142.504150.62616-10.67417-43.70818-100.66120132162480102812511=28.8935.05143.36751.3956-1007168-106.072950105011-4.80212-1.25130.498145.832157.03316-10.06817-79.37618-106.07220132162480184312581=21.4835.8342.1075-10.4196-104.7917168-103.949950105011-3.47212-12.44913-16.33214-14.88315-42.95116-91.21617-103.59518-103.94920132162480291612511=27.4335.12942.91956.116-104.8227168-103.86595010501116.286125.223134.379142.154151.03916-16.10417-88.16118-103.86520132162480413712631=20.48436.31542.75154.1586-103.2827158-103.28995.61050115.57120.75613-1.54414-50.47115-98.43416-103.28917-103.06618-103.26720132162480519611053.4308450222015463.430845022201547168-10.0999501050114.304128.586139.522147.183154.016167.21817-3.28218-10.09920132162480720212451=232.0334.49441.14554.0356-1007158-46.6639501050113.007126.8313-0.646142.14157.76816-6.50817-16.60318-46.66320132162480731112471=218.5534.73242.597518.3046-1007168-93.79995010501124.8951229.6351329.398145.887158.06165.87217-21.49118-93.79920132162480810611051.5339249372482361.53392493724823715820.45791.81050112.478124.33413-2.113148.4331520.457162.63417-1.77118-12.71920132162480853612531=24.8235.31743.9015-7.3586-108.3067168-109.263950105011-3.42412-10.57613-4.36514-14.00415-5.12416-48.25617-104.80918-109.26320132162481165912471=221.734.66441.0235-2.4416-1007168-59.85895010501112.57412-3.1413-6.69314-8.50515-12.9916-18.43217-18.47418-59.85820132162481219812611=20.83636.07843.82650.9876-104.167168-104.38495.61050112.904120.06913-0.33214-12.37615-94.42716-104.38417-104.1618-103.77920132162481257111055.8300509452819865.83005094528198716828.37590.023510501128.3751210.48137.597144.70715-0.799162.96517-2.7218-3.96420132162481374712511=27.1335.14743.695-2.7436-108.4347158-108.1529501050115.99412-2.70413-7.50714-2.93215-6.98516-18.68717-97.62418-108.15220132162481565912491=213.9934.85443.8055-4.0026-1007168-104.687950105011-8.639128.96813-5.713141.5215-9.19616-12.83317-45.6718-104.68720132162481570312471=218.9534.7224458.2386-1007168-102.91695010501116.992121.018132.196147.801155.5231614.90217-18.03618-102.91620132162481570612451=233.1434.4841.98452.4856-1007168-55.699501050112.976124.23131.771142.065153.32316-2.04617-8.98718-55.6920132162481757512531=24.6735.33142.15514.2536-110.2857168-106.07895010501112.3611210.81316.2141421.16515-1.57316-43.95217-102.63618-106.07820132162481827712581=21.7335.76241.273514.2886-112.3727168-107.61895010501119.514127.109135.809142.72915-41.97316-74.19817-106.60518-107.61820132162481841912591=21.2635.940.9485256-116.1487168-106.55695010501130.3771220.663130.04514-2.6715-47.04516-79.11817-106.03618-106.55620132162481960412511=27.1635.1454459.3156-86.5747168-90.30691610501119.3181214.679134.366143.901152.74116-11.35117-90.30618-79.9620132162482006912401=2103.5433.98540.55256-257168-20.33195010501136.8761213.48130.083148.099151.4971610.45217-10.3818-20.33120132162482394812671=20.18136.74243.425520.4696-104.7977168-104.93490.610501122.1781215.10813-50.36114-104.93415-104.28516-103.84717-104.51118-104.04220132162482397712481=215.834.8014459.5086-1007168-106.7049501050116.459127.341137.1171415.1511513.801166.42117-35.41318-106.70420132162482415612491=212.1734.91542.081512.7566-1007168-106.08995010501125.311210.2151313.8971412.354153.78916-8.07617-44.12418-106.089201321624824916110511.9809885025024611.98098850250247158-102.58395010501125.2481217.8661317.9861413.7921511.7931613.8951740.56918-102.58320132162482499812571=22.1735.6644456.0636-105.2787168-105.0739501050111.538123.225135.171413.75815-17.43716-102.99617-104.00618-105.07320132162482502112511=28.1435.08942.788510.846-1007158-104.5199501050118.5831213.437135.5251414.393158.48316-11.46117-79.74118-104.51920132162482568412441=242.634.37140.55256-1007168-35.72595010501117.6971223.4921326.18114-1.67715-0.98916-9.26117-3.20118-35.72520132162482660712551=22.9435.53243.3695-3.8626-104.5117168-104.305916105011-7.31612-1.32313-5.33914-1.51915-11.97916-83.94417-104.30518-104.01320132162482707812481=215.4934.8142.62453.3926-1007168-92.774950105011-0.378124.329132.258147.166153.89116-1.31417-36.918-92.77420132162482756512581=21.6935.77240.7515256-1007168-105.6595010501130.313129.81713-2.5314-14.27115-29.8116-58.5117-77.59218-105.6520132162482779311057.6835665702819867.68356657028198716816.4129501050119.332121.091136.64714-5.5041511.486167.7221714.2821816.41220132162483490812611=20.87936.05641.1635-7.7826-109.3647168-104.898950105011-5.19612-12.12613-16.61814-24.4815-56.15416-82.21817-104.40618-104.89820132162483593411052.813359022140562.8133590221405716813.992916105011-1.325122.881134.49914-1.921151.498164.3491713.99218-1.46520132162484113712621=20.70636.15142.1165-10.8746-106.267168-105.595916105011-11.82412-11.513-11.23314-30.44815-77.68416-105.10417-105.59518-104.86820132162665794612461=225.4634.59443.02356.7436-1007168-81.92795010501111.047120.566138.482147.788153.232168.86317-8.08218-81.9272013216266591051105-14.2031040191656-14.2031040191657168-36.79950105011-14.49612-10.1513-10.53614-11.10315-12.13716-4.64917-13.76318-36.79201321626660325110513.6860618591309613.6860618591309713827.36690.023510161127.3661211.4271312.404148.5361510.2531610.4651717.01820132162666042412501=210.8534.96543.8635-12.2076-1007158-103.802950105011-11.90712-6.50513-14.09914-9.40915-15.04316-15.90517-65.55518-103.80220132162666112612501=210.6834.9714457.0176-42.9837168-36.743916105011-1.1051216.1713-5.169140.24215-4.12816-4.73617-36.74318-21.01420132162666147012561=22.635.58542.0665-6.8136-107.0647168-104.8039501050113.43712-8.26713-14.49614-10.26215-22.22316-72.02817-103.5918-104.80320132162666218812521=26.2835.20242.5475-2.2196-1007148-103.699950105012-0.191135.42514-4.06315-13.44416-27.27317-85.68418-103.69920132162666248312511=27.3235.13542.184511.8696-1007168-105.96795010501118.9541213.1031315.779148.82815-3.63616-17.16717-77.99618-105.96720132162666319512491=213.5634.86843.3175-10.2536-1007168-96.564950105011-8.12912-5.59413-9.17214-16.91915-12.30416-11.14817-46.02418-96.56420132162666366412571=22.1735.66442.87950.5546-106.5817158-106.699950105011-2.337129.81113-2.675140.82815-23.62616-93.94817-105.85118-106.69920132162666383212541=23.8935.4142.6315256-108.9727168-107.69895010501123.6131228.7141326.8661426.1531511.97516-65.46517-106.39318-107.69820132162666420812541=24.3135.36641.871516.816-110.3437168-105.84695010501131.0761221.136137.006143.788153.08116-48.76317-100.03718-105.84620132162666478112741=20.041737.3841.824506-109.9887158-113.549950105011-17.26412-55.03913-101.82714-106.54115-107.62916-108.14217-110.05118-113.54920132162666680712571=22.1435.674456.0736-108.0687158-107.7295010501114.813128.7713-0.903145.2915-18.56616-105.82217-106.77518-107.7220132162666686412621=20.65736.18242.63251.6826-104.8597168-104.76595.610501111.75312-0.81813-10.28614-27.29315-94.32116-104.76517-104.31118-103.31720132162667014412481=216.0234.79542.3415-5.7146-1007168-90.39295010501110.58212-8.99713-10.27314-5.88215-6.01516-19.80917-31.52818-90.392201321626670293110518.1559524536133618.1559524536133716823.37191.810501122.4411222.0661318.4651420.8171523.3711615.3171718.922183.8520132162667140012491=211.6634.93342.998519.7666-1007168-105.7295010501122.2341219.491322.8941414.7841520.257167.95317-61.21218-105.7220132162672683011050.20355568826198660.2035556882619867158-28.23395010501119.4151211.2113-4.148141.86715-7.39116-4.57117-0.73918-28.23320132162672694612471=220.9134.6841.8695-4.5286-1007158-71.7249501050113.08512-9.03913-5.62514-6.40615-6.05816-5.87317-26.02218-71.72420132162672729912461=223.9634.62141.97159.8136-1007158-72.1619501050119.7141213.5831310.7521413.901151.055167.17717-16.31618-72.16120132162672980411059.2951793670654369.29517936706543715820.83290.023510501120.8321215.405138.818145.45156.638164.677179.33188.9820132162673023012551=23.1935.49642.24557.5746-108.1087158-105.9359501050115.107127.0471311.9144.80115-8.99416-68.84517-105.28518-105.93520132162673025511058.9671268463134868.96712684631348715813.68890.211050116.986128.0561313.6881410.236156.8661610.1571712.73418-2.94120132162673263312601=21.0135.99642.623513.1546-108.4847168-109.31995010501117.2461211.216139.20914-3.63115-78.05116-105.8817-107.90618-109.31920132164650123112481=217.7734.7543.03153.7246-1007158-94.32295010501113.0571211.916134.70914-3.03115-3.2616-3.02917-26.81618-94.32220132164719493512451=234.9234.45741.9154.2816-1007158-52.73895010501128.483124.871131.762141.28815-3.275162.09217-7.65318-52.73820132164719530812541=24.0635.3914458.0996-106.8527168-106.6391610501119.632122.214134.81145.653155.67816-70.74817-106.6318-106.2732013216471954011105-14.10946464538576-14.10946464538577168-22.152950105011-11.49412-5.75713-14.314-13.20815-14.60516-13.29917-18.06118-22.15220132164719812812511=28.2435.08443.38357.0236-107.3727168-106.89495010501110.267129.512134.055145.986154.80116-8.40617-89.78218-106.89420132164964098412641=20.40136.39741.449517.3796-108.0237158-107.05795010501127.909123.25213-8.94614-51.23915-93.72316-105.22317-105.61518-107.0572013216496413791105-15.73909854888926-15.73909854888927168-21.0219161050111.2512-19.11813-15.54414-17.08715-19.31516-14.41717-21.02118-20.66220132164964175211059.4762144088745169.47621440887451715813.77490.61050116.885126.6281313.3511413.7741510.942169.6521710.01918-0.35920132164964184912471=220.3334.69244516.7416-1007168-101.35895010501126.1621222.3611323.694147.7821513.938165.11317-9.15418-101.35820132164964309212561=22.5735.59445-24.3236-105.5777158-105.552950105011-28.02412-24.24813-25.15414-22.28815-26.3116-87.6617-105.10118-105.55220132164964499712571=2235.69944515.3316-105.9887168-105.4449501050119.8831212.4381317.071421.22315-22.62416-105.08417-105.01318-105.44420132164964564412461=225.6834.5942.3895256-1007148-76.86595010501126.2931245.8591323.211421.0581524.5411624.15917-4.04418-76.865201321649645652110520.6348171234131620.6348171234131714895.11890.023510501195.1181240.5631326.758146.8271515.8081615.54917-2.03218-3.17620132164964596112601=21.0135.996445-11.7326-104.6777168-104.702950105011-13.51312-11.99313-11.51714-13.02315-84.44716-104.4717-104.17718-104.7022013216496482061105-4.870202541351326-4.870202541351327168-8.719916105011-4.78512-6.93813-7.14114-7.781151.063160.03117-8.71918-4.6920132164964902112571=21.9335.714445-0.4636-104.9697168-104.3579161050111.5912-5.9713-0.793142.71215-28.29316-103.68217-104.35718-104.25120132164966589911058.4305114746093868.43051147460938716814.78690.61050115.41214.0461311.8511414.7861512.9241614.335177.0718-12.96720132164966602212461=225.7534.58942.6945-3.3156-1007158-75.394950105011-1.917125.37513-6.11514-11.952150.51916-6.93117-14.09218-75.39420132164966660112601=20.96236.01742.14956.5626-106.7177158-106.6919501050114.458128.192134.46614-13.25915-71.54716-102.35817-106.33218-106.69120132164966661912571=22.0535.68842.4510.3166-106.2287158-105.7739161050117.0511214.805137.175148.47715-27.14116-90.64517-105.77318-105.70220132164966779312761<20.023537.62944506-105.8697158-106.79590.21105011-40.86312-103.88113-106.79514-105.97815-105.57516-104.88517-106.07818-105.65420132164966869312561=22.6835.5724451.1146-106.6557158-106.4169501050111.17912-1.871130.208143.58515-6.58316-98.69917-105.48718-106.41620132164967016612531=25.2135.2834457.5046-105.1147168-105.44495010501111.995124.608134.006146.1471510.0416-42.70117-103.11418-105.44420132164967199512551=23.2635.48741.84558.8876-111.3557168-106.8395010501110.711126.1381313.232142.27815-11.52416-62.10917-105.86718-106.832013216496723511105-4.469422817230226-4.469422817230227158-15.231950105011-1.61312-9.18213-3.11214-2.49915-0.00816-0.35717-3.03818-15.23120132164967247812511=28.8235.055445256-108.3217148-106.75295010501114.6591245.1651344.7361429.3611530.1951612.58217-97.35418-106.75220132164967546712471=221.0734.67643.2452.5976-1007168-90.0589501050112.78912-1.047131.12140.848157.461163.74917-17.27818-90.058201321649713967110511.3935890197754611.3935890197754716825.9891.81050114.791213.8841312.1931421.6911525.981613.065179.47718-9.93220132164971648512521=27.0235.15443.84254.186-107.3697158-106.9949501050119.039124.242137.416144.94215-2.85516-16.22717-99.63218-106.99420132164971784012471=221.2234.673445-1.6266-1007168-104.0429501050117.71912-6.2913-5.55714-11.88315-3.737166.75717-11.03918-104.04220132164973595612451=230.4234.51741.9385-10.196-1007158-43.407950105011-18.33812-15.8713-9.06914-3.24415-7.46116-11.00117-18.75118-43.4072013216497369261105-10.78170299530036-10.78170299530037168-18.5099501050117.03512-3.26613-7.8114-16.97115-12.9216-18.32517-15.48818-18.5092013216498165451105-8.352737426757816-8.352737426757817168-18.46695010501112.81712-0.97713-10.47514-11.02515-6.3616-14.42517-17.9118-18.46620132164981784612461=225.9934.58540.9755-0.0186-1007158-51.77195010501110.816124.90113-3.81414-6.15315-10.29716-7.15317-23.6118-51.77120132164982340212491=211.8434.92741.613511.1416-1007168-85.1069501050113.5961213.9331311.0211410.0891514.51616-10.36117-48.69918-85.10620132164982360612441=243.4834.3624452.1726-1007158-46.82895010501113.43712-0.044132.12214-2.384150.14616-1.327172.08418-46.828201321649824379110512.5931100845337612.5931100845337715838.87395010501111.437124.048131.2781411.014155.3591610.251712.8291838.87320132164982445012501=210.2334.9942.33956.9796-1007168-94.5849501050116.543125.893136.555148.925155.36316-7.05417-62.78218-94.58420132164982537212461=224.5634.6141.5295-11.2946-1007168-57.954950105011-8.0112-16.07513-10.30614-6.4215-13.9816-18.72117-23.18318-57.9542013